The present invention relates to an amylase of a new type, an .alpha.-amylase-pullulanase enzyme active and stable at high temperatures, by means of which it is possible to produce maltose syrup from starch. This soluble enzyme is produced in the culture medium by certain Clostridium thermohydrosulfuricum strains when they are grown on a culture medium which contains dextrin or small-molecular soluble starch.
The glucose units of starch are linked to each other mainly by .alpha.-1,4-glucosidic linkages, forming long chains. In addition, starch has .alpha.-1,6-glucosidic linkages, there being such a linkage at each branching site of the chains.
.alpha.-Amylases cleave 1,4 linkages of starch at random along the chains, whereas .beta.-amylases cleave the same linkages at the non-reducing ends of the chains. Pullulanases, for their part, are debranching enzymes which cleave the 1,6-linkages at the branching sites.
Maltose syrups are prepared by hydrolyzing starch by means of plant .beta.-amylases or by allowing a saccharifying mold .alpha.-amylase to hydrolyze further a starch which has been liquefied using bacterial .alpha.-amylase. By both procedures a maltose syrup containing approximately 60% maltose is obtained. Maltose yields higher than this are obtained using .beta.-amylase together with pullulanase. Maltose syrups are used mainly in the candy and bakery industries, because of their typical mild sweet taste, low viscosity, low hygroscopicity, and high thermal stability.
It is advantageous for the starch hydrolysis process if the enzyme used is active and stable at high temperatures. .beta.-Amylases, mold .alpha.-amylases, and commercially available pullulanases of Klebsiella pneumoniae and Bacillus sp. cannot, however, be used at temperatures above 60.degree. C. Thus, a maltogenic enzyme with higher thermostability would be advantageous in the production of maltose syrup.
If several enzymes are used in the process, it is usually necessary to make compromises with respect to the activity requirements of the enzymes, or alternatively to use the enzymes in succession and to adjust the process in between. In terms of simplicity and economy it would be better if one single enzyme was sufficient for the process. For this reason the ideal enzyme used for the production of maltose syrup should possess liquefying, saccharifying and starch debranching activity.
Hyun and Zeikus (1985; J. Bacteriol. 49, 1168) studied the degradation of starch by Clostridium thermohydrosulfuricum strain E39 (ATCC 33223) isolated from the hot Octopus spring in Yellowstone National Park in the United States. This strain produced cell-bound pullulanase and glucoamylase but not .alpha.-amylase. The preparations obtained from strain E39 hydrolyzed starch, producing glucose, without maltose, maltotriose or maltotetraose being observed as intermediate products.